Skip to main content
. 2018 Mar 27;9(2):e02229-17. doi: 10.1128/mBio.02229-17

FIG 4 .

FIG 4 

Stress-specific Hog1 outputs are differentially affected by C156, C161, and C271. (A) Impact of C156S, C161S, and C271S mutations upon the redox status of Hog1 following 10 min of exposure to 2.5 mM DPTA-NONOate, as revealed by AMS gels (see the legend to Fig. 3A). The differences in Hog1 masses between wild-type cells and HOG1C→S mutants reflect changes in disulfide bond formation in Hog1 in the absence and presence of stress. WT, Ca2226; C156S, Ca2222; C161S, Ca2224; C156/161S, Ca2225; C271S, Ca2216 (Table S1). For the C156/161S columns, the wild-type controls were from the same blot as that showing the lanes with (+) and without (−) DPTA-NONOate and treatment with NEM, DTT, and AMS in Fig. 3A. (B) Impact of C156S, C161S, and C271S mutations upon the nitrosative-, oxidative-, and osmotic-stress sensitivity of C. albicans. Strains were spotted in 10-fold serial dilutions onto plates containing no stress (control), 1 M NaCl, 5 mM H2O2, 5 mM NaNO2, or 2.5 mM DPTA-NONOate. All figure panels for the wild type, null mutant, and C156S and C161S single and double mutants were derived from the same set of plates. The C271 mutant, which displayed no obvious stress phenotype, was compared to the wild-type control in a separate experiment. (C) Impact of C156S, C161S, and C271S mutations upon the phosphorylation dynamics of Hog1 phosphorylation following exposure of cells to 2.5 mM DPTA-NONOate or 5 mM H2O2. Western blotting was performed on cell extracts prepared at the times indicated (in minutes). Phosphorylated Hog1 (P-Hog1) was detected by probing the blots with phospho-p38 antibody, and for loading controls, the blots were reprobed for total Hog1 and actin. The results are indicative of three independent replicate experiments, and the data for the individual HOG1C156S and HOG1C161S mutants are shown in Fig. S4. (D) Impact of C156S, C161S, and C271S mutations upon the Hog1-mediated induction of nitrosative (YHB1)- and oxidative (TRR1)-stress genes in response to 5 mM H2O2 or 2.5 mM DPTA-NONOate for 10 min. The strains are as described for panel A. The fold induction of the YHB1 and TRR1 transcripts was measured by qRT-PCR, relative to the internal ACT1 mRNA control, and by normalizing to the levels of these transcripts in the absence of the stress. Means and standard deviations are shown for three independent replicate experiments. *, P < 0.05.